CRISPR enzymes and systems

Patent metadata

Field	Value
Publication number	US-12410468-B2
Application number	US-202017034553-A
Country	US
Kind code	B2
Filing date	Sep 28, 2020
Priority date	Jun 18, 2015
Publication date	Sep 9, 2025
Grant date	Sep 9, 2025

Abstract

The invention provides for systems, methods, and compositions for targeting nucleic acids. In particular, the invention provides non-naturally occurring or engineered DNA-targeting systems comprising a novel DNA-targeting CRISPR effector protein and at least one targeting nucleic acid component like a guide RNA. Methods for making and using and uses of such systems, methods, and compositions and products from such methods and uses are also disclosed and claimed.

First claim

What is claimed is:

1. A modified eukaryotic cell or progeny thereof, wherein the modified eukaryotic cell is obtained ex vivo by treating a wild-type cell with a CRISPR-Cpf1 system comprising (a) a Cpf1 protein or a polynucleotide encoding the Cpf1 protein and (b) a Cpf1 guide targeting a genomic sequence in the wild-type cell positioned 3′ of a T-rich protospacer adjacent motif (PAM) recognized by the Cpf1 protein, or a polynucleotide encoding the Cpf1 guide,
wherein a CRISPR complex formed by the Cpf1 protein and the Cpf1 guide binds to and cleaves the genomic sequence, and wherein the modified eukaryotic cell comprises an insertion, deletion, or substitution of one or more nucleotides in the genomic sequence compared to the wild-type cell;
wherein the Cpf1 guide comprises, from 5′ to 3′, a direct repeat sequence linked to a guide sequence; wherein the direct repeat sequence comprises, from 5′ to 3′, a stem left region, a loop region, and a stem right region; wherein the stem left region comprises UCUAC, CCUAC, UCCAC, or UCUGC, and the stem right region is complementary to the stem left region and comprises GUAGA, GUAGG, GUGGA, or GCAGA.

2. The modified eukaryotic cell or progeny thereof of claim 1 , wherein the modified eukaryotic cell is obtained by treating the wild-type cell with a CRISPR-Cpf1 system comprising the Cpf1 guide and the Cpf1 protein.

3. The modified eukaryotic cell or progeny thereof of claim 1 , wherein the modified eukaryotic cell is obtained by treating the wild-type cell with a CRISPR-Cpf1 system comprising a ribonucleoprotein (RNP) complex formed by the Cpf1 guide and the Cpf1 protein.

4. The modified eukaryotic cell or progeny thereof of claim 1 , wherein the modified eukaryotic cell is obtained by treating the wild-type cell with a vector encoding the Cpf1 guide and the Cpf1 protein.

5. The modified eukaryotic cell or progeny thereof of claim 4 , wherein the vector is a viral vector.

6. The modified eukaryotic cell or progeny thereof of claim 5 , wherein the viral vector is an adenoviral vector, a lentiviral vector, or an adeno-associated viral vector.

7. The modified eukaryotic cell or progeny thereof of claim 1 , wherein the modified eukaryotic cell is obtained by treating the wild-type cell with a CRISPR-Cpf1 system comprising the Cpf1 guide and an mRNA encoding the Cpf1 protein.

8. The modified eukaryotic cell or progeny thereof of claim 1 , wherein the CRISPR-Cpf1 system further comprises a template DNA for homology-directed repair of the cleavage.

9. The modified eukaryotic cell or progeny thereof of claim 1 , wherein the CRISPR-Cpf1 system is delivered into the wild-type cell by microinjection, electroporation, sonoporation, biolistics, calcium phosphate-mediated transfection, cationic transfection, liposome transfection, dendrimer transfection, heat shock transfection, nucleofection transfection, magnetofection, lipofection, impalefection, optical transfection, proprietary agent-enhanced uptake of nucleic acids, and delivery via liposomes, immunoliposomes, virosomes, or artificial virions.

10. The modified eukaryotic cell or progeny thereof of claim 1 , which is a human cell.

11. The modified eukaryotic cell or progeny thereof of claim 10 , which is a hematopoietic cell or a lymphocyte.

12. The modified eukaryotic cell or progeny thereof of claim 10 , which is a hematopoietic CD34+ stem/progenitor cell, a natural killer cell, a cytotoxic T lymphocyte, a regulatory T lymphocyte, or a tumor-infiltrating lymphocyte, a pluripotent stem cell.

13. The modified eukaryotic cell or progeny thereof of claim 10 , which is a pluripotent stem cell.

14. The modified eukaryotic cell or progeny thereof of claim 1 , wherein the genomic sequence is associated with a genetic disease or disorder.

15. The modified eukaryotic cell or progeny thereof of claim 14 , wherein the genetic disease or disorder is a blood disease or disorder.

16. The modified eukaryotic cell or progeny thereof of claim 14 , wherein the genetic disease or disorder is sickle cell anemia or beta-thalassemia.

17. The modified eukaryotic cell or progeny thereof of claim 14 , wherein the genetic disease or disorder is a cancer.

18. The modified eukaryotic cell or progeny thereof of claim 1 , wherein the modified eukaryotic cell or progeny thereof has altered expression of one or more of PD1, CTLA4, TRAC, TRBC, B2M, and MHC2TA, compared to the corresponding wild-type cell.

19. The modified eukaryotic cell or progeny thereof of claim 1 , wherein the Cpf1 protein is from Francisella tularensis 1, Prevotella albensis , Lachnospiraceae bacterium MC2017 1 , Butyrivibrio proteoclasticus , Peregrinibacteria bacterium GW2011_GWA2_33_10, Parcubacteria bacterium GW2011_GWC2_44_17, Smithella sp. SCADC, Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237, Leptospira inadai, Lachnospiraceae bacterium ND2006 , Porphyromonas crevioricanis 3 , Prevotella disiens , or Porphyromonas macacae.

20. The modified eukaryotic cell or progeny thereof of claim 1 , wherein the Cpf1 protein is Francisella novicida U112 Cpf1 (FnCpf1), Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1), or Lachnospiraceae bacterium ND2006 (LbCpf1).

21. The modified eukaryotic cell or progeny thereof of claim 1 , wherein the Cpf1 protein is FnCpf1 and the PAM is TTN, where Nis A, C, G or T; or wherein the Cpf1 protein is AsCpf1 or LbCpf1 and the PAM sequence is TTTV, where Vis A, C or G.

22. The modified eukaryotic cell or progeny thereof of claim 1 , wherein the loop comprises UGUU, UAUU, UCUU, UUUU, or UAAGU.

23. The modified eukaryotic cell or progeny thereof of claim 22 , wherein the Cpf1 guide further comprises AAUU or AUU upstream of the stem left region, and a uracil followed by a guide sequence downstream of the stem right region.

24. A pharmaceutical composition comprising the modified eukaryotic cell or progeny thereof of claim 1 .

25. A non-human animal or livestock comprising the modified eukaryotic cell or progeny thereof of claim 1 .

Assignees

• Broad Inst Inc
• Massachusetts Inst Technology
• Harvard College
• Massachusetts Instuitute Of Tech
• President And Fellows Of Harvard Colleage

Inventors

• Zhang Feng
• Zetsche Bernd
• Gootenberg Jonathan S
• Abudayyeh Omar O
• Slaymaker Ian

Classifications

• C12Q1/6816

  characterised by the detection means (C12Q1/6804 takes precedence) · CPC title

• C12N15/113

  Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)} · CPC title

• C12N9/22 Primary

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title

• C12N2800/80

  Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites · CPC title